Aim: The study aimed at investigating urinary neopterin, a marker of cellular immune response, and urinary N-acetyl-β- D-glucosaminidase (NAG), a marker of tubular damage, as noninvasive means to differentiate between acute tubular necrosis (ATN) and rejection in initially nonfunctioning (INF) human renal transplants. Methods: Seventy-two renal transplant patients were studied. Forty-five of them experienced an uncomplicated early posttransplant course, 27 patients suffered from INF. Twenty-two patients experienced ATN, 5 patients had a total of six biopsy-proven rejections. The NAG activity was measured by a colorimetric assay, neopterin by high-performance liquid chromatography. Receiver operating characteristics (ROC) analysis was applied to compute diagnostic performance and an optimal discriminating threshold. Results: Demographic characteristics (age, gender, cold and warm ischemia periods, HLA mismatches) and posttransplant urinary NAG and neopterin excretions did not differ between ATN and rejection groups. Both urinary NAG and neopterin excretions were lower in the control group (NAG 1.8 ± 1.0 U/mmol urinary creatinine; neopterin 270 ± 126 nmol/mmol urinary creatinine; mean ± SD) as compared with the ATN group (NAG 12 ± 10 U/mmol, p < 0.001 vs. control group; neopterin 303 ± 195 nmol/mmol, n.s.) and the rejection group (NAG 7 ± 8 U/mmol, p < 0.01; neopterin 508 ± 419 nmol/mmol, p < 0.01). The ratio of urinary neopterin to NAG excretion (uNNR; dimension nmol neopterin/U NAG activity) increased during rejections as compared with ATN (139 ± 74 vs. 50 ± 38 nmol/U, p < 0.01). The area under the ROC curve for uNNR was 0.88 ± 0.07 (p < 0.001). Applying a ROC-estimated optimal discriminator of uNNR (80 nmol/U), 16 patients with ATN and all six rejection episodes were classified correctly. Conclusion: The uNNR provides a noninvasive means to aid in the differential diagnosis of rejection and ATN in INF human renal transplants.